Microfluidic device technologies, also referred to as lab-on-a-chip technologies, have been proposed for a number of different applications in various fields. In the field of biology, for example, microfluidic devices may be used to carry out cellular assays. In addition, microfluidic devices have been proposed to carry out separation techniques in the field of analytical chemistry.
Generally, microfluidic devices may be used to separate the components of a fluid sample using either of two techniques: capillary electrophoresis or chromatography. Capillary electrophoresis involves the separation of molecules based on differences in the electrophoretic mobility of the molecules. Typically, microfluidic devices employ a controlled application of an electric field to induce fluid flow and/or to provide flow switching. In order to effect reproducible and/or high-resolution separation, a fluid sample “plug” (a predetermined volume of fluid sample) must be controllably injected into a capillary separation column or conduit. For fluid samples containing high molecular weight charged biomolecular analytes, such as DNA fragments and proteins, microfluidic devices containing a capillary electrophoresis separation conduit that is a few centimeters in length may be effectively used to carry out separation of small sample plugs having a length on the order of micrometers. Once injected, high sensitivity detection, such as laser-induced fluorescence, may be employed to resolve a separated fluorescent-labeled sample component.
For samples containing analyte molecules with low electrophoretic mobility differences, such as those containing small drug molecules, the separation technology of choice is generally chromatography. Chromatographic separation occurs when a mobile phase carries sample molecules through a chromatography bed (stationary phase) where sample molecules interact with the stationary phase surface. The velocity at which a particular sample component travels through a chromatography bed depends on the component's partition between mobile phase and stationary phase. Microfluidic devices that incorporate a liquid chromatographic functionality have been described in U.S. Ser. No. 09/908,231. These microfluidic devices may employ integrated mechanical valve technologies, such as those described in U.S. Ser. No. 09/908,292, for sample introduction and to reduce the volume of “dead space” in the microfluidic devices.
There are many chromatographic techniques known in the art. See e.g., Kutter et al. (1998), “Solvent-programmed microchip open-channel electrochromatography,” Anal. Chem. 70:3291-3297. For example, in reverse phase liquid chromatography, where the stationary phase offers a hydrophobic surface and the mobile phase is usually a mixture of water and an organic solvent, the least hydrophobic component moves through the chromatography bed first, followed by other components, in order of increasing hydrophobicity. In other words, the chromatographic separation of sample components may be based on hydrophobicity.
In isocratic liquid chromatography, the content of the mobile phase is constant throughout the separation. Gradient liquid chromatography, on the other hand, requires the content of the mobile phase to change during separation. Gradient liquid chromatography not only offers high resolution and high-speed separation of wide ranges of compounds, it also allows injection of large sample volumes without compromising separation efficiency. During the initial period when the sample is introduced, the mobile phase strength is often kept low, and the sample is trapped at the head of the liquid chromatography column bed. As a result, interfering moieties, such as salts, are washed away. In this regard, gradient liquid chromatography is suited to analyze fluid samples containing a low concentration of analyte moieties.
Typically, pressurizing means are employed to provide flow through packed columns in liquid chromatography. Such pressurizing means typically include pumps that are designed for optimal performance at a certain flow rate range, generally between about 50 μL/min to about 1 mL/min. To generate a gradient of a selected mobile-phase component within a mobile phase, two pumps may be employed to pump two fluids, each fluid containing a different concentration of the selected mobile-phase component. The fluids are mixed to form the mobile phase and introduced into the column. By varying the relative flow rate of the pumps, a concentration gradient of the selected mobile-phase component may be formed within the mobile phase flowing through the column. However, the quality of the gradient generated by this technique is limited by the performance of the pumps. In some cases, a gradient may require a fluid flow rate that lies outside the capability of the pumps. This limitation is particularly pronounced when microbore liquid chromatography columns are employed, because the required mobile-phase flow rate through the columns is extremely low.
Use of conventional chromatographic equipment with microfluidic devices for separating the components of a fluid may present other technical problems. For example, in order to obtain a smooth gradient, conventional liquid chromatography systems employ a pressure damper as well as a mixer. The pressure damper and the mixer require a certain volume of fluid for proper operation. This volume is associated with a delay time, i.e., the time it takes for a mobile phase to reach the liquid chromatography column after mixing. The delay time can be calculated as the quotient of the delay volume over the flow rate. For example, the combined volume of the damper and the mixer in a conventional liquid chromatography system is about 0.3 mL to about 0.5 mL. This translates to a delay time of about 0.3 minutes to about 0.5 minutes at a flow rate of 1 mL/min. However, if a microfluidic device is constructed for operation at a flow rate of less than about 1 μL/min, the delay time increases to about 300 minutes to about 500 minutes. Such a long delay time renders microfluidic device-based gradient liquid chromatography impractical.
To overcome this limitation, commercial low flow rate liquid chromatography pumps typically employ a split flow design. In this design, pumps move mobile phase at a high flow rate, but only a portion of the mobile phase is delivered to the separation column. The remainder of the mobile phase is diverted into a waste stream. This adds to the cost of operation because typically less than 1% of the mobile phase is actually used for separation.
In addition, the concentration of the selected mobile-phase components delivered to the column changes as gradient liquid chromatography is carried out. In most cases, the viscosity of the mobile phase also changes. This tends to change the pressure profile within the column. To compensate for such pressure profile changes, an electronic flow control unit that includes a flow meter and a variable flow resistor is used to control commercial chromatography pumps. As mobile phase is delivered to the column, the flow meter measures the flow rate of the mobile phase to provide feedback control over the variable flow resistor. Nevertheless, current flow sensor technologies are incapable of accurately measuring flow rates of less than 1 μL/min, especially in gradient mode, and are therefore unsuitable for use in microfluidic devices that are employed in gradient chromatography.
Thus, there is a need for integrated microfluidic device technology that allows control and generation of a gradient of a selected mobile-phase component within a small volume of mobile phase in order to separate the components of a fluid sample. In particular, there is a need for an improved microfluidic device that employs a smooth gradient at low flow rates, especially in flow rate ranges of less than 1 μL/min.